CN108777947A

CN108777947A - Plant and preparation method thereof with blue series pattern

Info

Publication number: CN108777947A
Application number: CN201780018398.3A
Authority: CN
Inventors: 野田尚信; 中山真义; 道园美弦; 本乡智; 间龙太郎; 胜元幸久
Original assignee: Suntory Holdings Ltd
Current assignee: Suntory Holdings Ltd
Priority date: 2016-03-31
Filing date: 2017-03-13
Publication date: 2018-11-09
Anticipated expiration: 2037-03-13
Also published as: WO2017169699A1; US11299742B2; CO2018011157A2; US20200325486A1; JPWO2017169699A1; JP6758622B2; CN108777947B; CA3018690A1

Abstract

Technical task to be solved by this invention is to provide the complicated blue expression mechanism that need not be proposed in the past or the method reappeared its technology, and make the plant with blue series pattern by more easy blue expression control technology.Make 3 ' and 5 ' positions of anthocyanidin B rings by glycosylation delphinidin type anthocyanidin and flavone glycoside or flavonol glycosides the coexisting into the cell in floral organs such as the petals of plant as copigment.

Description

Plant and preparation method thereof with blue series pattern

Technical field

The present invention is the production method of the plant with blue series pattern, is related to 3 ' and 5 ' the position quilts so that anthocyanidin B rings Glycosylation delphinidin type anthocyanidin coexists in plant cell with copigment (flavone glycoside or flavonol glycosides) and is characterized Method and so that anthocyanidin B rings 3 ' and 5 ' positions by glycosylation delphinidin type anthocyanidin and copigment (flavone glycoside or Person's flavonol glycosides) coexist in the cell the plant with blue series pattern being characterized or its from grow either he grow offspring or Their nutrition propagule, a part (especially cut-flower) for plant or its processed goods (especially cut-flower processed goods), group It knits or cell.

Background technology

Chrysanthemum, rose, carnation, lily etc. are flowers important in worldwide industry.However, in such main flowers In, there is no the wild species etc. of blue series pattern in interfertile sibling species, in previous crossbreeding, mutational breeding The kind with blue series pattern is made to have difficulties.About this present inventor in recent years by by 5 ' H of Canterburybells F3 ' and butterfly 5 ' GT of beans A3 ' carry out channel genes, have successfully produced more blue the case where than individually importing 5 ' H of CamF3 ' (patent document 1) With being that Violet-Blue95, Violet-Blue97 or Blue100 etc. are referred to as in Royal Horticultural Association (RHSCC) Really " blue chrysanthemum " (patent document 2) of blue pattern.However, in rose, lily, carnation, dahlia, iris etc. It in other main flowers, does not produce with the kind with the blue degree of " blue chrysanthemum " same degree, and is groping Regulation and Blue Flowers facture.

Gene when carrying out pattern change, imported by genetic recombination for colored blueization is based in nature The opinion of the blue expression mechanism of Regulation and Blue Flowers present in boundary carrys out selection.Due to the anthocyanidin accumulated in most of Regulation and Blue Flowers Basic framework be delphinidin type, so be responsible for its biosynthesis 3 ', 5 '-oxidizing ferment of flavonoids gene for Regulation and Blue Flowers Make the change (patent document 3) for being imported into realize pattern.On the other hand, even if being delphinidin type anthocyanidin, powder is expressed Red, reddish violet, purple, bluish violet flower largely exist in nature.It is reported that except flower in colored blue expression mechanism Other than emerald green element type anthocyanidin, it is also necessary to Intramolecular association (intramolecular be total to color), Interpolymer Association (intermolecular color altogether) and metal from The interaction of son, metal complex formed, rising of pH etc. (non-patent literature 1) in vacuole.

When being accumulated in petal by 2 or more aromatic organic acids and sugar-modified polyamides delphinidin type anthocyanidin, Blue is expressed by Intramolecular association.From rough gentian (patent document 4,5), the delphinium for expressing blue pattern by the mechanism Glycosyltransferase gene, aromatic acyl are reported in (patent document 6, non-patent literature 2), butterfly beans (patent document 2,7) etc. The relative enzyme gene of transferase gene etc..Have for blue expression as when being coexisted with anthocyanidin in Interpolymer Association (color altogether) The copigment of effect, it is also reported that participate in C- glycosyl flavones biosynthesis enzyme gene (non-patent literature 3), can in rose into The gene (patent document 8) of the synthesis of row flavonoids.Reported in the interaction with metal ion in tulip iron from Sub to participate in, aluminium ion participates in laurustinus, and the gene that transport metal ion is reported in the vacuole of anthocyanidin accumulation is (non-special Sharp document 4).In addition, in the formation of metal complex, it is believed that in order to be formed necessary to complex with metal ion for controlling The 7- glycosidase genes of the glycosylation mode of manufacture-yellow ketone report (non-patent literature 5) in radix scutellariae, arabidopsis, 4 ', 7- glycosidases Gene is reported in happiness woods careless (patent document 9) etc..Other also reported that participating in Intramolecular association, Interpolymer Association or metal matches Close object formation by be bonded on the 3 of anthocyanidin hydroxyls sugar further by sugar-modified glycosyltransferase gene, It is bonded to the acyl transferase gene for the acyl group that organic acid is shifted on the sugar of anthocyanidin.Specify makes importing base in main flowers Because of effective promoter, terminator in the expression of petal.It is therefore contemplated that import participate in the genes of these blue expression mechanisms from And it is feasible in theory to function.

The flowers with blue series pattern are made by the method for genetic engineering, are had for carnation and rose Commercially available, pattern is purple (RHSCC hue groups：Purple), bluish violet (Purple-Violet, Violet) has hyacinthine (Violet-Blue), there is no made the Blue flower of blue (Blue) pattern in addition to " blue chrysanthemum " (patent document 2) Make.Therefore in order to make various blue expression mechanisms (non-patent literature 1) in the flowers of carnation, lily or rose etc. It reappears, has attempted the isolated channel genes to import and combine them of related gene, but could not simultaneously produce Regulation and Blue Flowers.This Be due to the polyamides anthocyanidin of the color development of most of responsible Regulation and Blue Flowers, metal complex it is complicated, in their synthesis It is middle to need a large amount of gene.Example from the isolated a series of gene for being responsible for its blue expression of the Regulation and Blue Flowers of nature is limited 's.It has accumulated to carry out the polyamides anthocyanidin biosynthesis that the gene group needed for channel genes and blueization only has rough gentian Genome.If even if by assembling for whole quiding genes needed for blue expression, due to the importing and control of lots of genes Become complicated, so the making of Regulation and Blue Flowers becomes difficult.

Existing technical literature

Patent document

Patent document 1：No. 2010/122849 bulletin of International Publication No.

Patent document 2：No. 2017/002945 bulletin of International Publication No.

Patent document 3：No. 2010/069004 bulletin of International Publication No.

Patent document 4：No. 1996/025500 bulletin of International Publication No.

Patent document 5：No. 2006/046780 bulletin of International Publication No.

Patent document 6：No. 2011/016260 bulletin of International Publication No.

Patent document 7：No. 2007/046148 bulletin of International Publication No.

Patent document 8：No. 2008/156211 bulletin of International Publication No.

Patent document 9：No. 2012/096307 bulletin of International Publication No.

Patent document 10：No. 2002/086110 bulletin of International Publication No.

Patent document 11：No. 2000/044907 bulletin of International Publication No.

Patent document 12：No. 1994/003606 bulletin of International Publication No.

Patent document 13：No. 2013/157502 bulletin of International Publication No.

Patent document 14：No. 2006/105598 bulletin of International Publication No.

Non-patent literature

Non-patent literature 1：Nat.Prod.Rep.(2009)26：884

Non-patent literature 2：Plant Cell Physiol.(2015)56：28

Non-patent literature 3：J Biol.Chem.(2009)284：17926

Non-patent literature 4：Plos one(2012)7：E43189, Genes to Cells (2013) 18：341

Non-patent literature 5：Planta(2000)210：1006, Biosci.Biotechnol.Biochem. (2006) 70：1471

Non-patent literature 6：Saito et al., (1983) A cyanidin glycoside giving scarlet Coloration in plants of the Bromeliaceae., Phytochemistry 22：1735-1740

Non-patent literature 7：Andersen et al., The anthocyanins, in Flavonoids, Chemistry, Biochemistry and applications, Edited by Andersen, O.M.&Markham, K.R., Taylor& Francis, pp.472-537 (2006)

Non-patent literature 8：Shimizu-Yumoto et al., (2012) Slantingly cross loading sample system enables simultaneous performance of separation and mixture to detect Molecular interactions on thin-layer chromatography.J.Chromatogr.A, 1245：183- 189

Invention content

Technical task to be solved by this invention is to provide based on the blue table entirely different with previous theory, technology Blue series pattern (the RHS chromatographies standard the 5th edition for having and being unable to get in the past is made up to control technology：Violet-Blue groups/ Blue groups and/or hue angle：230 °~290 °) plant production method.

Present inventor has made intensive studies and experiment has been repeated in order to solve the above problems, has as a result obtained as follows Astonishing opinion：Even if without the modification that aromatic organic acid carries out, by making 3 ' and 5 ' positions of anthocyanidin B rings by glucosides The delphinidin type anthocyanidin of change, which coexists with copigment (flavone glycoside or flavonol glycosides) in plant cell, can also make tool There is the plant of blue series pattern, so as to complete the present invention.

That is, the present invention is as follows.

[1] a kind of method, for the production method of the plant with blue series pattern, which is characterized in that make anthocyanidin B rings 3 ' and 5 ' positions are by glycosylation delphinidin type anthocyanidin and copigment (flavone glycoside or flavonol glycosides) in the intracellular total of plant It deposits.

[2] method according to 1, which is characterized in that the flavone glycoside be selected from cyanidenon glucosides, quercetin glucosides, Apiolin glucosides, acacetin glucosides and combination thereof.

[3] method according to 2, which is characterized in that the cyanidenon glucosides is cyanidenon 7- malonyl glucose Glycosides, cyanidenon 7- glucosides, cyanidenon 7,3 '-diglucoside, cyanidenon 8-C- glucosides, cyanidenon 6- C- glucosides or their derivative.

[4] method according to 2, which is characterized in that the quercetin glucosides is quercetin 7- malonyl glucose Glycosides or derivatives thereof.

[5] method according to 2, which is characterized in that the apiolin glucosides is apiolin 7- glucosides, apiolin 7- rues Fragrant glucosides, apiolin 8-C- glucosides, apiolin 6-C- glucosides or their derivative.

[6] method according to 2, which is characterized in that the acacetin glucosides is acacetin 7- rutinosides or it spreads out Biology.

[7] method according to 1, which is characterized in that the flavonol glycosides be selected from Kaempferol glucosides, quercetin glycoside and it Combination.

[8] method according to 7, which is characterized in that the Kaempferol glucosides is Kaempferol 3- glucosides or derivatives thereof.

[9] method according to 7, which is characterized in that the quercetin glycoside is Quercetin 3- glucosides, Quercetin 3- (6 "-malonyl) glucoside, Quercetin 3- rutinosides or their derivative.

[10] method according to any one of 1~9, which is characterized in that 3 ' and 5 ' positions of the anthocyanidin B rings are glycosylation Delphinidin type anthocyanidin be selected from delphinidin 3- (6 "-malonyl) glucoside -3 ', 5 '-diglucosides, that is, ternatin C5,3,3 ', 5 '-tri-glucose of delphinidin glycosides, that is, preternatin C5 and combination thereof.

[11] method according to any one of 1~10, which is characterized in that 3 ' and 5 ' positions of the anthocyanidin B rings are by glucosides The delphinidin type anthocyanidin of change is with the flavone glycoside with 1：1~1：10 amount ratio coexists.

[12] method according to any one of 1~11, which is characterized in that pH in the vacuole of the plant is 5.2~ 6.4。

[13] method according to any one of 1~12, which is characterized in that the plant is rose, lily, carnation, big Beautiful flower, iris or chrysanthemum.

[14] a kind of plant or its from growing or he grows offspring, which is characterized in that be the plant with blue series pattern, in plant 3 ' and 5 ' positions of intracellular anthocyanidin B rings be total to flavone glycoside or flavonol glycosides by glycosylation delphinidin type anthocyanidin It deposits.

[15] plant according to 14 or its from growing or he grows offspring, which is characterized in that the flavone glycoside is selected from sweet-scented osmanthus Careless element glucosides, quercetin glucosides, apiolin glucosides, acacetin glucosides and combination thereof.

[16] plant according to 15 or its from growing or he grows offspring, which is characterized in that the cyanidenon glucosides is wood Rhinoceros grass element 7- malonyls glucoside, cyanidenon 7- glucosides, cyanidenon 7,3 '-diglucoside, cyanidenon 8-C- glucosides, cyanidenon 6-C- glucosides or their derivative.

[17] plant according to 15 or its from growing or he grows offspring, which is characterized in that the quercetin glucosides is five Sunkatol No. 1 7- malonyl glucosides or derivatives thereof.

[18] plant according to 15 or its from growing or he grows offspring, which is characterized in that the apiolin glucosides is celery Plain 7- glucosides, apiolin 7- rutinosides, apiolin 8-C- glucosides, apiolin 6-C- glucosides or they spread out Biology.

[19] plant according to 15 or its from growing or he grows offspring, which is characterized in that the acacetin glucosides is gold Silk tree element 7- rutinosides or derivatives thereof.

[20] method according to 14, which is characterized in that the flavonol glycosides be selected from Kaempferol glucosides, quercetin glycoside and Combination thereof.

[21] method according to 20, which is characterized in that the Kaempferol glucosides is Kaempferol 3- glucosides or its derivative Object.

[22] method according to 20, which is characterized in that the quercetin glycoside is Quercetin 3- glucosides, Quercetin 3- (6 "-malonyl) glucoside, Quercetin 3- rutinosides or their derivative.

[23] plant according to any one of 14~22 or its from growing or he grows offspring, which is characterized in that the cyanine 3 ' and 5 ' positions of plain B rings are selected from delphinidin 3- (6 "-malonyl) glucoside -3 ' by glycosylation delphinidin type anthocyanidin, 5 '-diglucosides, that is, ternatin C5,3,3 ', 5 '-tri-glucose of delphinidin glycosides, that is, preternatin C5 and their group It closes.

[24] plant according to any one of 14~23 or its from growing or he grows offspring, which is characterized in that the cyanine 3 ' and 5 ' positions of plain B rings are by glycosylation delphinidin type anthocyanidin and the flavone glycoside or flavonol glycosides with 1：1~1： 10 amount ratio coexists.

[25] plant according to any one of 14~24 or its from growing or he grows offspring, which is characterized in that the plant Vacuole in pH be 5.2~6.4.

[26] plant according to any one of 14~25 or its from growing or he grows offspring, which is characterized in that the plant For rose, lily, carnation, dahlia, iris or chrysanthemum.

[27] a kind of nutrition propagule, a part for plant, tissue or cell, which is characterized in that from according in 14~26 Any one of them plant or its from growing or he grows offspring.

[28] a kind of cut-flower or the processed goods made by the cut-flower, which is characterized in that from the plant described in any one of 14~27 Object or its from growing or he grows offspring.

According to the present invention, it is not only chrysanthemum, in other main flowers such as rose, lily, carnation, dahlia, iris In can also make be unable to get in the past have blue series pattern (RHS chromatographies standard the 5th edition：Violet-Blue groups/Blue groups And/or hue angle：230 °~290 °) kind.Especially, including the flavone glycoside or flavones as copigment appropriate It, can be only by the way that the method for the glycosylation such simplicity in 3 ' and 5 ' positions of anthocyanidin B rings be made " Regulation and Blue Flowers " during alcohol glucosides is spent.

Description of the drawings

Fig. 1 is the anthocyanidin included in the petal of the genetic recombination chrysanthemum of the chrysanthemum and blue series pattern of host：a) Cyanidin type anthocyanidin (A1-A4)；B) delphinidin type anthocyanidin (A5-A10).

The result for a) indicating that blue Chrysanthemum Petal extracting solution is unfolded by Cross TLC methods of Fig. 2 (under fluorescent lamp).B) it indicates The result that blue Chrysanthemum Petal extracting solution is unfolded by Cross TLC (under UV light (365nm)).Bands of a spectrum A includes B rings by glucosides The anthocyanidin (ternatin C5 and preternatin C5) of change.Color development is that the arrow of grey is also used in the part of blue in bands of a spectrum A Head indicates.Show that the bands of a spectrum C1 of yellow fluorescence intersects under w light in the arrow head part of grey.Color development is purple in bands of a spectrum A Bands of a spectrum C2 (dotted line) dark under w light also intersects in part.A is separately included in C1 and C2 becomes blue, purple copigment object Matter.C) structure of the C1 and C2 of identification and d) are indicated.Arrow indicates HMBC.

Fig. 3 a) indicate that the extinction of the visible domain of blue is composed.In Ternatin C5 (TC5；A8 the knot of mixing Lt7MG (C1) in) Fruit is with making with the amount of TC5 ratio from 1：1 increases to 1：5,1：10, it is characteristic in 600-620nm ranges in blue petal Absorbance rise, while maximum absorption wavelength is also to long wavelength side displacement, and becomes extinction spectrum identical with blue petal.b) Indicate the extinction spectrum of the visible domain of purple/bluish violet.Risen by adding absorbances of the C1 near 570nm in A5, is shown It shows to compose identical spectral pattern with the extinction of purple petal.C) Mg of the addition A8 equivalent in addition to C1 and C2 is indicated²⁺Ion or The Fe of 1/10 equivalent of A8³⁺The extinction of visible domain when ion is composed.

Fig. 4 indicates the B cyclohexanol glycosidation anthocyanidin under various pH conditions (pH4.0,4.6,5.0,5.6,5.8,6.0,6.6,7.0) (TC5) it is composed with the color of flavone glycoside (Lt7MG) mixed liquor and extinction.

Fig. 5 indicate B cyclohexanol glycosidation anthocyanidin (TC5) with various flavone glycosides (Lt7MG, Lt7G, Lt3 ' 7G, Tr7MG, Ap7G, Ap7RG, Aca7RG) mixed liquor extinction spectrum.

Fig. 6 indicates the anthocyanidin (Pg3MG, Cy3MG, Dp3MG3 ' G, TC5) and flavones that there are various B rings to embellish a design The extinction spectrum (left figure) of the mixed liquor of glucosides (Lt7MG).Indicate the mixed liquor of anthocyanidin and flavone glycoside under various conditions The suction of (Lt7MG+TC5 (pH5.6), Lt7MG+pTC5 (pH5.6), Tr7MG+pTC5 (pH5.6), Lt7MG+pTC5 (pH5.8)) Spectrum (right figure).

Fig. 7 indicate B cyclohexanol glycosidation anthocyanidin (TC5) with various flavone glycosides (Lt7OMG=Lt7MG (C1), Lt8CG, Lt6CG, Ap8CG, Ap6CG) mixed liquor extinction spectrum.

Fig. 8 shows B cyclohexanol glycosidation anthocyanidin (TC5) and flavone glycoside (Lt7MG) or various flavonol glycosides (Km3G, Qu3G, Qu3MG, Qu3RG) mixed liquor extinction spectrum.

Specific implementation mode

The present invention is the production method of the plant with blue series pattern, is related to 3 ' and 5 ' the position quilts so that anthocyanidin B rings Glycosylation delphinidin type anthocyanidin coexists in plant cell with copigment (flavone glycoside or flavonol glycosides) and is characterized Method.

Anthocyanidin is the pigment group being widely present in plant, it is known that present red, purple, blue pattern.According to work For the hydroxyl value of the B rings at the anthocyanidin position of aglycone, be classified as pelargonidin, Cyanidin and delphinidin 3 are System.Chromophore is aglycon moiety, and orange red, Cyanidin presentation red is presented in pelargonidin, and delphinidin presentation is purplish red Color.Be known as the 3 ' positions of B rings of the primary pigments of blue Chrysanthemum Petal, 5 ' positions by glycosylation anthocyanidin with it is not glycosylation Anthocyanidin compare, the maximum absorption wavelength of extinction in acid condition spectrum to short wavelength side displacement (non-patent literature 6), into One step reports pattern (non-patent literature 7) of the expression with red degree when B rings are accumulated by glycosylation anthocyanidin in petal.

Present inventor this time has found by making 5 ' H genes of Canterburybells F3 ' with 5 ' GT genes of butterfly beans A3 ' simultaneously in Chrysanthemum Petal Blue chrysanthemum (patent document 2) made by middle expression includes delphinidin 3- (6 "-malonyl) glucose as primary pigments Glycosides -3 ', 5 '-diglucosides (ternatin C5, TC5) include to take off the third two as ternatin C5 as micro pigment 3,3 ', 5 '-tri-glucose of delphinidin glycosides (preternatin C5, pTC5), delphinidin 3- (3 ", 6 "-two malonyl of acyl group body Base) glucoside -3 ', 5 '-diglucosides, delphinidin 3- (6 "-malonyl) glucoside -3 '-glucoside (Dp3MG3 ' G) and Cyanidin 3- (6 "-malonyl) glucoside -3 '-glucoside (Cy3MG3 ' G).As blue Maximum absorption wavelength is 511nm in the HPLC analyses of the ternatin C5 of the main anthocyanidin of chrysanthemum in acid condition, With Cyanidin 3- (6 "-malonyl) glucosides (Cy3MG) of the main anthocyanidin of red, pink as script Maximum absorption wavelength 518nm is compared, further to short wavelength side displacement.This means that although ternatin C5 than script arrow Che Jusu type anthocyania pigments are redder, but color development is blue in the petal of chrysanthemum.Anthocyanidin B rings as Ternatin C5 3 ' positions and 5 ' position hydroxyls the example of blue expression is carried out in colored petal organ etc. by glycosylation red cyanidin so far The present is not reported.

By making the substance of anthocyania pigment and flavones, flavonols, organic acid ester, tannins etc. coexist, with they into Row intermolecular interaction so that express the color with blue degree sometimes.The phenomenon is referred to as color altogether, and (copigment acts on, is intermolecular Color, Interpolymer Association altogether), cause the substance of the phenomenon to be referred to as copigment (copigment).Not only have in total color and causes indigo plant The bathochromic effect that color table reaches, also heavy colour effect, the effect for improving colour stability.Therefore, present inventor speculates in blue chrysanthemum Hua Zhong, ternatin C5, preternatin C5, delphinidin 3- (3 ", 6 "-two malonyl) glucoside -3 ', 5 '-two Portugals 3 ' and 5 ' positions of the anthocyanidin B rings of polyglycoside etc. pass through the copigment with plant endo by glycosylation delphinidin type anthocyanidin Matter interaction etc. is to make petal expression blue.

In present specification, " 3 ' and 5 ' positions of anthocyanidin B rings are by glycosylation delphinidin type anthocyanidin " is as long as with Huang Ketoside or flavonol glycosides coexist and blue are presented, and are just not particularly limited, for example, can enumerate ternatin C5, Preternatin C5 etc..Additionally, it is believed that finding the delphinidin 3- (3 ", 6 "-two of accumulation in Blue Gene recombinates Chrysanthemum Petal Malonyl) glucoside -3 ', 5 '-diglucosides also coexist with flavone glycoside or flavonol glycosides and blue are presented.

Flavones is one kind of organic compound, is the cyclic ketone of flavan derivatives, is existed mainly as glucosides in plant. Flavones refers to chemical formula C in the narrow sense₁₅H₁₀O₂, the compound of molecular weight 222.24,2,3-, bis- dehydrogenation flavane -4- ketone, the Huang of broad sense Ketone (flavonoids) is one of classification of flavonoids, using flavones structure as basic framework in flavonoids, and is not had at 3 further The flavonoids of hydroxyl is classified as " flavones ".In the specification of the present application, " flavone glycoside " is the flavones of broad sense, this means that belonging to In the glucosides of the derivative of flavonoids.As flavone glycoside, cyanidenon glucosides, quercetin glucosides, apiolin sugar can be enumerated Glycosides, acacetin glucosides, and it's not limited to that.Cyanidenon glucosides, quercetin glucosides, apiolin glucosides, acacetin Glucosides comprise in addition cyanidenon, quercetin, apiolin, acacetin derivative glucosides.As cyanidenon sugar Glycosides, such as cyanidenon 7- (6 "-malonyl) glucoside (Lt7MG), cyanidenon 7- glucoside (resedas can be enumerated Glycosides), cyanidenon 7,3 '-diglucoside, cyanidenon 8-C- glucosides (orientin), cyanidenon 6-C- glucosides (homo-orientin) or their derivative as quercetin glucosides, such as can enumerate quercetin 7- (6 "-malonyl) Portugal Polyglycoside (Tr7MG) or derivatives thereof as apiolin glucosides, such as can enumerate apiolin 7- glucoside (cosmos Glycosides), apiolin 7- rutinosides (different Rhoifolin), apiolin 8-C- glucosides (Vitexin), apiolin 6-C- glucose Glycosides (isovitexin) or their derivative additionally as acacetin glucosides, such as can enumerate acacetin 7- rutinosides (linarin) or derivatives thereof.

Flavonols are one of the classification of flavonoids, have 3-hydroxyflavone (3- hydroxyl -2- phenyl chromene -4- ketone) skeleton.? Exist mainly as glucosides in plant.In the specification of the present application, " flavonol glycosides " meaning belongs to the derivative of flavonols Glucosides.As flavonol glycosides, Kaempferol glucosides, quercetin glycoside, Myrica rubra can be enumerated, and it's not limited to that. Kaempferol glucosides, quercetin glycoside, Myrica rubra comprise in addition Kaempferol, Quercetin, myricetin derivative glucosides. As Kaempferol glucosides, such as Kaempferol 3- glucosides, Kaempferol 3- rutinosides or their derivative can be enumerated, in addition As quercetin glycoside, such as Quercetin 3- glucosides, Quercetin 3- (6 "-malonyl) glucoside, quercitrin can be enumerated Plain 3- rutinosides or their derivative.

Flavones synthesizes in the petal of many plants such as chrysanthemum, carnation, African Chrysanthemum, is accumulated as glucosides, but in rose Flavones is not detected from petal, or there is also the plants for only accumulating denier in rare, lily or Lisianthus etc..In order in this way Plant petal in so that flavones is synthesized and make the glucosides accumulate, can be used flavone synthetase gene.For example, can be from various plants Kind come clone coding as ketoglutaric acid dependence dioxygenase flavones synzyme I (FNSI) gene (patent document 10) and Encode the gene (patent document 11) of the flavones synthetase II (FNSII) as NADPH dependent form Cytochrome P450s.Therefore, By the way that these genes are carried out channel genes, Huang can not also be synthesized from the plant for detecting flavone glycoside in petal in rose etc. Ketone, the glucosides can accumulate (patent document 8) in petal.In addition, flavonols is in many plants such as rose, lily or Lisianthus It synthesizes in petal and is accumulated as glucosides, but there is also chrysanthemum, carnation, African Chrysanthemums etc., and flavonols to be not detected from petal Or only accumulate the plant of denier.In order to synthesize flavonols in the petal of such plant and its glucosides be made to accumulate, can be used Flavonol synthase gene (FLS).Huang of the coding as ketoglutaric acid dependence dioxygenase can be cloned from various plant species The gene (patent document 12) of keto-alcohol synzyme.Therefore, by the way that the gene is carried out channel genes, even if in chrysanthemum etc. not from flower Valve, which detects in the plants of flavonol glycosides, can also synthesize flavonols, its glucosides can be made to be accumulated in petal.About in the present invention Workable plant is not particularly limited, such as rose, lily, carnation, dahlia, iris or chrysanthemum can be enumerated etc., it can There are the Lisianthus of method of quiding gene, cyclamen, forget-me-not, orchid, African Chrysanthemum, dendrobium, tulip, India using report The various plants such as certain herbaceous plants with big flowers pigment, petunia, Bowring cattleya.

In the plant of such flavone glycoside accumulation, by by 3 ' and 5 ' position glucosides of the intracellular anthocyanidin B rings Change, 3 ' and 5 ' positions of anthocyanidin B rings can be made to be coexisted with flavone glycoside by glycosylation delphinidin type anthocyanidin.Anthocyanidin B rings The glycosylation of 3 ' and 5 ' positions can be by will be from 3 ', the 5 '-O- glucosyl transferase genes of anthocyanidin (5 ' GT of CtA3 ') of butterfly beans It imported into plant to realize (patent document 2).In addition, can also by by comprising 3 ', 5 ' positions by the plant of glycosylation anthocyanidin Sundries official, tissue realize 3 ', 5 ' position glycosyltransferase gene cloning of anthocyanidin.In addition, can also make 5 ' GT of CtA3 ' and come (patent document 2) is co-expressed from 3 ', 5 '-oxidase gene of flavonoids (5 ' H of CamF3 ') (patent document 13) of Canterburybells.

In the present state-of-the technology, in order to by gene transfered plant and make the gene constitutive character or tissue specificity table It reaches, it can be by the way that well known to a person skilled in the art method such as agrobacterium co-cultivation, binary vector method, electroporation, PEG methods, particles Marksmanship etc. carries out.

By make 3 ' and 5 ' positions of anthocyanidin B rings by for glycosylation delphinidin type anthocyanidin and flavone glycoside typical case with 1：1~1：10, preferably 1：5~1：10, most preferably from about 1：10 amount ratio coexists, and blue can be presented.

Its color development is controlled the intermolecular total color of anthocyania pigment and anthocyania pigment and copigment due to pH.Bear color development Pigment accumulation the intracellular organelles such as vacuole in pH can be inserted directly into electrode to measure, pass through and measure the tissues such as petal and squeeze The pH of juice can also determine general value.It is carried out in tissues such as petals to be made in the spending of the plant of blue expression, even if in flower When the pH of the green plain vacuole coexisted with copigment accumulation is not appropriate for expressing by the blue that total color carries out, by making adjusting pH Gene function can also make vacuole pH optimize.The pH of vacuole is by being present in vacuole's type H+-ATP enzymes (V- of vesicular membrane ATPase) with the function of vacuole's type proton pump of vacuole's type H+-pyrophosphatases (V-PPase) as acidity, lead to It crosses and promotes or inhibit the function of these genes that pH (patent document 14) is adjusted.In addition, pH rises to 7.7 from 6.6 when blooming, flower In sky blue morning glory of the color from purple variation for blue, it was recently reported that coding is by along with the transport by potassium ion into vacuole And proton ion is discharged to make pH rise sodium ion-hydrogen ion reverse transport protein (NHX) gene (non-patent literature 1) it, may be adjusted to pH in the vacuole to be more easy to make blue color development.It is 5.2~6.4 for pH typical case in the vacuole of presentation blue Left and right, preferably 5.4~6.2 or so, most preferably 5.6~6.0 or so are not limited to the range as long as blue can be presented.

In turn, the invention further relates to by the available plant with blue series pattern of the above method or its from growing or He grows the cut-flower or the processed goods made of the cut-flower (especially cut-flower processed goods) of offspring.In this as cut-flower processed goods, Including embossing, preserved flower, dried flower, the resin seal product etc. for using the cut-flower, but not limited to this.

Embodiment

[embodiment 1：That is accumulated in the petal of blue chrysanthemum has been bonded the flower of sugar in 3 ' position of B rings on the hydroxyl of 5 ' positions The identification of emerald green element glucosides]

5 ' H of the F3 ' (5 ' H of CamF3 ' for having imported and having made from Canterburybells will be used；Patent document 13；GenBank accession number：FW570877) with coding 3 ', 5 '-glycosidase of anthocyanidin from butterfly beans CtA3 ' 5 ' GT (patent documents 2； GenBank accession number：AB115560 obtained by) Agrobacterium of the binary vector pB423 co-expressed is converted The main anthocyanidin of " big flat " transformant being displayed in blue analyzed with LC-MS.The pattern of the chrysanthemum ligule petal used When carrying out comparison colour examining under fluorescent light using Royal Horticultural Association (RHSCC) the 5th edition, has and be equivalent to Violet- The blue pattern of Blue 97, Blue100.It calculates the L in CIEL*a*b* color specification systems^*It is worth (lightness), a^*It is worth (red green), b^*Value (yellow blue color) measures the average value of 3 times or more values using CD100 colour meters (Yokogawa Meters&Instruments), with this When calculating hue angle (Hue angle) based on value, the value of the hue angle for the blue for indicating 230-290 ° is shown.LC-MS points Analysis using ACQUITY UPLC BEH C18 chromatographic columns (1.7 μm, 2.1i.d.x 100mm；Waters) pass through in 35 DEG C ACQUITY UPLC carry out classification separation, are gone here and there by ACQUITY UPLC photodiode arrays (PDA) detector and ACQITY Join quadrupole mass spectrometer (TQD) (Waters) to detect.It uses 1% aqueous formic acid (solvent A) in the mobile phase of UPLC and contains The acetonitrile (solvent B) of 1% formic acid.The flow velocity of mobile phase is 0.1ml/ minutes, carries out 0-5%B (0-5 minutes), 5-35%B (5- 20 minutes), the gradient elution of 35%B (20-25 minutes).By PDA detect 200-800nm, obtained at 360nm flavonoids with The chromatography of acyl group quinine acids, obtains the chromatography of anthocyanidin at 530nm.Mass spectrometric analysis condition is as follows：ESI just from Subpattern；Capillary voltage, 3.5kV；Orifice potential, 45V；Ion source temperature, 150 DEG C；Desolventizing temperature, 350 DEG C；Desolventizing Gas flow rate flow rate, 500L/h；Taper hole gas flow rate, 50L/h；Collision energy, 6V, 20V；Quality measurement range, 180- 1080m/z.It is A7 and A8 that the result of the chromatography of resolved detection wavelength 530nm, which is main anthocyania pigment,.In addition, as micro color Element has detected 8 Anthocyanins (A1-6, A9-10, Fig. 1) using A3 as representative.

The result that A8 is carried out to LC-MS/MS analyses is to have obtained m/z=875 [M] as precursor ion⁺, as product from Son has obtained m/z=713 (- glucose), 627 (- glucose-malonyls), 465 (- 2 × Glc- malonyls), 303 (- 3 × Glc- malonyls；Delphinidin).In addition, carrying out isolated purifying from petal and measuring¹H-NMR, NOESY collection of illustrative plates (table 1) and height Resolution ESI-TOF-MS collection of illustrative plates (table 2), to be accredited as ternatin C5 (Fig. 1).A7 is carried out to the knot of LC-MS/MS analyses Fruit is to have obtained m/z=789 [M] as precursor ion⁺, 627 (- Glc), 465 (- 2 × grapes have been obtained as product ion Sugar), 303 (- 3 × glucose)), because it is found that consistent with the retention time of the HPLC of the acid hydrolysis products of A8 and collection of illustrative plates, so It is considered preternatin C5 (Fig. 1).In blue petal LC-MS points will be carried out as the A3 detected by micro anthocyanidin The result of analysis is to have obtained m/z=773 [M] as precursor ion⁺.From petal it is isolated purifying and carry out NMR parsings (¹H-NMR、 NOESY) and precision mass is analyzed, and is accredited as Cy3MG3 ' G (Fig. 1, table 1, table 2).

Table 1

Isolated anthocyanidin¹H (600MHz) NMR spectra data

A3：In DMSO-d6+TFA, A8：In CD₃In OD+TFA

Table 2

[embodiment 2：3 ' the position of B rings included in petal has been bonded the delphinidin glucosides of sugar on the hydroxyl of 5 ' positions The relationship of ratio and blue color development]

By import in the pink kind " SEI ARABELLA " for being used as resupinate woodbetony leaf or root (Decora) flower pattern 5 ' H of CamF3 ' and 5 ' GT of CtA3 ' parse the expression of the pattern of obtained transformant system and the composition, quiding gene of anthocyanidin.Cyanidin Type anthocyanidin is that the flower of main anthocyanidin is unrelated with the glucosides of 3 ' positions, although showing the powder close to the pattern of host with purple It is red.Delphinidin type anthocyanidin is the flower of main anthocyanidin compared with host, and blue tone becomes strong and purple or indigo plant is presented Color, 3 ' positions and 5 ' positions are higher by the ratio of glycosylation anthocyanidin, and blue tone is stronger.It was found that the ratio of delphinidin type anthocyanidin Example and 3 ' positions related to the expression quantity of 5 ' H of CamF3 ' and 5 ' positions are by the expression of 5 ' GT of the ratio of glycosylation anthocyanidin and CtA3 ' Amount is related.It can be seen from the above result that as 3 ', blue is born in 5 ' positions by the A7 and A8 of the delphinidin type anthocyanidin of glucosyl Color development.

[embodiment 3：It is bonded on the hydroxyl of 5 ' positions in 3 ' position of B rings in the solution of the delphinidin glucosides of sugar and Chrysanthemum Petal In extinction spectrum difference]

The extinction spectrum of chrysanthemum ligule petal is used into the spectrophotometer UV-2450 for being mounted with integrating sphere auxiliary equipment ISR-2200 (Shimadzu Seisakusho Ltd.) is measured.The absorption maximum of the visible light region of the pink petal of host species " SEI ARABELLA " (λ vismax) is 556nm, and the λ vismax of purple recombinant are the 561nm for being more likely to long wavelength domain.The λ of blue recombinant Vismax is the 573nm for being further intended to long wavelength domain, and 615nm nearby has characteristic acromion region.In HPLC acidity Under the conditions of measurement in, the λ vismax of the primary pigments A1, A2 of the host of pink colour pattern are 518nm, are recombinated relative to this purple The λ vismax of the primary pigments A5 of body are 527nm.On the other hand, the λ vismax as the main anthocyanidin A8 of blue recombinant The 511nm for being more likely to short wavelength domain is comparably with A1, A2.Respective main anthocyanidin is dissolved in as petal squeezeding juice pH PH5.6 acetate buffer solution when, the λ vismax of A1 are 533nm.The λ vismax of A5 are 535nm, and shoulder is found near 570nm Peak region.The λ vismax of A8 are 561nm, and acromion region is observed near 590nm.A8 has following feature：Although in acidity Under the conditions of compared with other anthocyanidin shortwave strong point have λ max, under mildly acidic conditions λ vismax have greatly to long wavelength side Displacement to color development be bluish violet.Due to any petal λ vismax compared with the λ vismax of each main anthocyanidin into One step is in long wavelength domain, so presumption shows the copigment of bathochromic effect in the pattern, especially blue color development of chrysanthemum Substance participates in.

[embodiment 4：It is coexisted with sugared delphinidin glucosides has been bonded on the hydroxyl of 5 ' positions in 3 ' position of B rings to which color development is The exploration of the copigment substance of blue]

Use the main flower of Cross TLC methods (non-patent literature 8) pair and the transformant of the chrysanthemum " big flat " with blue petal The copigment substance of green element interaction is explored.In a cold or frozen state by the blue petal about 200mg of genetic recombination chrysanthemum It crushes, 10% aqueous acetic acid, 500 μ l are to extract ingredient included in petal for addition.Use cellulose TLC glass plates Extracting solution is intersected and is opened up into line tilt according to (non-patent literature 8) is had been reported by (100 × 100mm, Merck company).It will The TLC plates opened up use developing solvent BAW (n-butanols：Acetic acid：Water=4：1：2 (v/v/v)) it is unfolded at room temperature.By expansion Plate air-dries, under fluorescent light under (Light Box NEW5000INVERTER, Fuji color) and UV light (254/360nm, CSN-15AC, Cosmo Bio) observation, while being shot with digital camera.The Rf values of anthocyan are ternatin C5 (A8)： 0.15；preternatin C5(A7)：0.11；Cy3MG3'G(A3)：0.30.

There is the red part of the presentation for thinking to detach with copigment in the evolute of anthocyanidin, exist simultaneously and think and auxiliary color The part (Fig. 2 a) that purple and blue is presented that element coexists.When observing under w light, yellow fluorescence is sent out in the part of blue Bands of a spectrum intersect, and in purple part, dark bands of a spectrum intersect (Fig. 2 b).Due to the C1 as the copigment included in each bands of a spectrum And C2 coexists with anthocyanidin, it is thus regarded that contributing to the expression of blue.C1 and C2 are extracted from TLC plates and pass through UPLC-MS/MS The result of analysis is that [M+H] can be obtained respectively⁺=535 with [M+H]⁺=551.

The peak of C1 and C2 with molecular weight 535 and 551 are purified by blue Chrysanthemum Petal extract.In addition by anthocyanidin A7, A8 and A3 are also purified, are isolated.

[embodiment 5：The purifying of copigment and the anthocyanidin glucosides for being bonded sugar on the hydroxyl of 5 ' positions in the position of 3 ' position of B rings/3 ' With structure determination]

The blue chrysanthemum ligule petal of acquisition is in -80 DEG C of preservations.The freezing petal of about 2.5kg is crushed in liquid nitrogen, in about 5.9L 10% aqueous formic acid in dipping to carry out the extraction of ingredient.Relative to the petal after extraction, the 10% of 6L is reused Aqueous formic acid extracts.After extracting solution is filtered using strainer (100 mesh), 10 minutes are centrifuged to obtain with 3,000rpm Supernatant.Supernatant is injected into the chromatographic column filled with Diaion HP-20 resins (Mitsubishi's chemical conversion) 3L.By chromatographic column with 9L's After the cleaning of 0.1% aqueous formic acid, the copigment for being adsorbed in resin is carried out again with anthocyanidin using the methanol containing 0.1% formic acid Elution.After eluent is concentrated, the residue for being freeze-dried obtained 14.2g is dissolved in 20% acetonitrile containing 0.1% formic acid Aqueous solution.By the sample of dissolving by YMC-Pack ODS A (S-15 μm, 50mm i.d. × 250mm) chromatographic column with flow velocity It 30ml/ minutes and uses 20% acetonitrile solution containing 0.1% formic acid as solvent, is detected at 360nm thus into one Step carries out classification separation.To include anthocyanidin A7 (preternatin C5), A8 (ternatin C5) and A3 (Cy3MG3 ' G) Component 1 includes 2 (C2 of copigment；Tr7MG component 2) includes 1 (C1 of copigment；Lt7MG component 3) is concentrated, is freezed respectively After drying, it is re-dissolved in 10% acetonitrile solution containing 0.1% formic acid.Component 1 is by separately including A3 and A7's and A8 Component the preparation HPLC (flow velocity 30ml/ minutes, the Detection wavelength that use 35% methanol containing 0.1% formic acid as solvent 530nm) carry out classification separation.The preparation HPLC of A3, A7 and A8 by using 20% methanol containing 0.5% formic acid as solvent (flow velocity 28ml/ minutes, Detection wavelength 530nm) further classification separation come it is isolated.

C1 and C2 by using 35% methanol containing 0.1% formic acid as solvent preparation HPLC (flow velocity 30ml/ minutes, Detection wavelength 280nm) carry out classification separation come it is isolated.By isolated copigment with anthocyanidin concentration, freeze-drying to obtain Powder (C1, the Lt7MG of yellow：201.5mg；C2,Tr7MG：52mg) with wine-colored powder (A8, ternatin C5：52mg； A7, preternatin C5：7mg；A3,Cy3MG3'G：9.8mg).

Copigment C1 and C2 are dissolved in DMSO-d6.Anthocyanidin A8 and A3 are dissolved separately in 10% (v/v) trifluoroacetic acid (TFA)-weight methyl alcohol mixed liquor and TFA- dimethyl sulfoxide (DMSO)s-d6 (DMSO-d6) mixed liquor.Copigment¹H-NMR、¹³C-NMR、 HMBC, HMQC, COSY each collection of illustrative plates, anthocyanidin¹H-NMR and NOESY collection of illustrative plates passes through Nuclear Magnetic Resonance JNM-ECZ600R/S1 (JEOL Ltd., Japan) is measured.¹H with¹³The resonant frequency of C is respectively 600.17MHz and 150.91MHz.

The high de-agglomeration energy quality analysis (HR-MS) of the copigment and anthocyanidin of purifying on Agilent1200LC by connecting ESI-TOF-MS (JMS-T100LP, Jeol) is met to carry out.Sample is dissolved in methanol with the concentration of 0.01mg/ml and is injected 10-20μl.Analysis condition is as follows：Solvent, methanol；Needle voltage, 2.2kV；1 voltage of hole, 85V；2 voltage of hole, 10V；Ring is saturating Mirror voltage, 25V；Peak tube voltage, 2kV；Flows of dry gases, 1L/ minutes, spray gas flow, 0.5L/ minutes；Desolventizing temperature Degree, 250 DEG C；1 temperature of hole, 80 DEG C.

The high resolution mass spectrum of copigment 1 (C1) can detect m/z values 535.10457 (M+H)⁺、557.10887(M+Na)⁺, With the molecular formula C from Lt7MG₂₄H₂₃O₁₄(535.10878)、C₂₄H₂₂O₁₄The uniform quality that Na (557.09725) is calculated.Copigment The high resolution mass spectrum of 2 (C2) can detect m/z values 551.10341 (M+H)⁺、573.09189(M+Na)⁺, with the molecule from Tr7MG Formula (C₂₄H₂₃O₁₅(551.10369)、C₂₄H₂₂O₁₅Na (573.08564)) calculate uniform quality.

By HR-MS collection of illustrative plates,¹H-NMR with¹³The one-dimensional NMR collection of illustrative plates of C-NMR and the two-dimentional NMR spectrum analysis of HMBC, HMQC, really The structure for determining C1 is Lt7MG, and the structure of C2 is Tr7MG (Fig. 2 c and d, table 2 and 3).

Table 3

Flavone glycoside¹H (600MHz) and¹³C (150MHz) NMR spectra data

Copigment (C1 and C2) is in DMSO-d6

[embodiment 6：It is being tried with copigment by the way that sugared delphinidin glucosides will be bonded on the hydroxyl of 5 ' positions in 3 ' position of B rings The blue petal extinction spectrum that mixing is carried out in pipe is built again]

First, anthocyanidin included in blue Chrysanthemum Petal and flavones are analyzed by HPLC, is quantitative.In anthocyanidin Flower is used from Detection wavelength 530nm, in flavones from the value of the HPLC peak areas of the obtained chromatographies of Detection wavelength 360nm The standard curve of emerald green element 3- glucosides (anthocyanidin) and cyanidenon (flavones) calculates the chemical combination object amount (nmol/ of unit petal mg).As a result, 3 '-and 5 '-positions of blue petal are by the total and copigment C1 of the anthocyanidin A7-A9 of glucosyl (Lt7MG) and the molar ratio average out to 1.0 of C2 (Tr7MG)：1.7：0.4.In addition, mole of anthocyanidin total amount and flavones total amount Ratio average out to 1：5.Based on above-mentioned ratio, it is 1 to make the ratio of the flavones relative to anthocyanidin：1~1：10 is mixed to make Close liquid.

Then, after the ligule petal of about 5g being crushed in liquid nitrogen, extraction is to obtain squeezeding juice in distilled water 15ml. Squeezeding juice is added in 15ml pipes, is centrifuged 10 minutes in 10 DEG C with 8,000rpm, by the pH of supernatant with being mounted with 9611-10D The pH meter D-71 (hole field makes institute) of pH electrodes is measured.Pattern builds experiment used as " big flat " transformant again The acetate buffer solution of the pH5.6 of the average pH of petal squeezeding juice carries out.By the Lt7MG and Tr7MG of the copigment (flavones) of purifying It is dissolved in dimethyl sulfoxide (DMSO) (DMSO).By the ternatin C5 (A8), Cy3MG3 ' G (A3), delphinidin 3- as anthocyanidin (6 "-malonyl) glucoside (Dp3MG) (A5), Cy3MG (A1) are dissolved in distilled water.It is purified from the lilac petal of butterfly beans Dp3MG.By Cyanidin 3- glucosides (Funakoshi) and malonyl-CoA (SigmaAldrich) uses from butterfly beans The thick enzyme of petal extraction synthesizes, purifies Cy3MG.

100mM acetate buffer solutions (pH5.6) (96-78 μ l) are added in 1.5ml miniature tubes, add the flavones DMSO of 10mM Solution (2-20 μ l) simultaneously makes it equably dissolve.Then, the anthocyanidin aqueous solution (2 μ l) for adding 10mM, by the anti-of uniform dissolution The 100 μ l injection black wall cuvettes of ultramicron (optical path length 10mm, Shimadzu Seisakusho Ltd.) of liquid are answered, UV-2450 spectrophotometers (island is used Tianjin makes institute) measure extinction spectrum.Make the ratio of the C1 relative to A8 from 1：1 becomes 1：10 are added, with C1 ratios Rise, maximum absorption wavelength and acromion are to long wavelength side displacement, and the absorbance of maximum absorption wavelength rises (Fig. 3 a).Relative to A8 add 5 equivalents more than C1 when, maximum absorption wavelength near 570nm, acromion region become 600-620nm, show with The identical collection of illustrative plates of blue petal, while the tone enhancing (Fig. 3 a) of blue.Additionally, it was found that there is also indigo plants identical with C1 in C2 Color expression effect.If showing, the blue that Chrysanthemum Petal can be achieved there are the C1 or C2 of 5 equivalents of A8 point is expressed.In addition, passing through C1 is added in A5, the absorbance near 570nm rises, and shows and composes identical spectral pattern (figure with the extinction of purple petal 3b).Result prompt also has identical copigment to participate in the color development of purple/bluish violet.

In order to investigate influence of the metal ion to blue expression, the addition for having carried out magnesium ion and iron ion is tested.It will make For the magnesium acetate (Mg (OAc) of divalent magnesium salts₂) distilled water is dissolved in the concentration of 10mM.In addition, by the sulphur as trivalent iron salt Sour iron ammonium (III) (NH₄Fe(SO₄)₂) distilled water is dissolved in the concentration of 1mM.The 2 μ l additions of each aqueous metallic ions are existed Including adding anthocyanidin after the acetate buffer liquor of flavones, the extinction spectrum of the reaction solution of 100 μ l of final quantity is measured.As a result, In addition to C1 and C2, by Mg²⁺Ion is with A8 equivalent or by Fe³⁺Ion is added with 1/10 equivalent of A8, and result is extinction spectrogram case Do not change (Fig. 3 c).It is therefore shown that metal ion has neither part nor lot in, the total color of B cyclohexanol glycosidation anthocyanidin and flavone glycoside becomes chrysanthemum The main reason for colored blue is expressed.

[embodiment 7：The total color of the delphinidin glucosides and flavone glycoside of sugar has been bonded on the hydroxyl of 5 ' positions in 3 ' position of B rings In the influence that color development is brought of various pH conditions]

The pH of the petal squeezeding juice of the incubation system or kind of various chrysanthemums and transformant is measured, as a result its value is 5.6 ~6.1 or so amplitude.Therefore, it uses first and is mixed into 0.2M disodium hydrogen phosphates aqueous solution and 0.1M aqueous citric acid solutions The McIlvaine buffer solutions of pH5.6,5.8,6.0, investigation pH draw the total colour response of B cyclohexanol glycosidation anthocyanidin and flavone glycoside The influence of the blue expression risen.B cyclohexanol glycosidation anthocyanidin uses A8 (ternatin C5), flavone glycoside to use C1 (Lt7MG). The McIlvaine buffer solutions (88 μ l) for being adjusted to each pH, the DMSO solution (10 of addition 10mM C1 are added in 1.5ml miniature tubes μ l) and equably dissolve.Then, the 100 μ l of reaction solution of the A8 aqueous solutions for being added to 10mM (2 μ l) uniform dissolution are injected into super In micro black wall cuvette (optical path length 10mm, Shimadzu Seisakusho Ltd.), extinction is measured using UV-2450 spectrophotometers (Shimadzu) Spectrum.As a result, compared with pH5.6 the maximum absorption wavelength of pH5.8 to long wavelength side (about 600nm) displacement, and then for pH6.0 when Absorbance near 600nm rises (Fig. 4).Thus the pH in the vacuole of petal is prompted to become 5.6, higher 5.8 or 6.0 etc. In kind system plants kind, and pH passes through as under 5.8 or 6.0 cultivation keeping shipping conditions in vacuole So that B cyclohexanol glycosidation anthocyanidin is coexisted with flavone glycoside can realize that more bright-coloured blue is expressed.

PH has each from 4 or so to 7 or so of morning glory etc. etc. of laurustinus etc. in the vacuole that anthocyanidin is accumulated in the spending of plant Kind various kinds.Therefore, various pH conditions are next investigated to color development is brought caused by total color influence.In 3 ', 5 '-glycosylation cyanine Ternatin C5 is used in element, uses C1 (Lt7MG) in the flavone glycoside of copigment, with 1：5 amount ratio is mixed.? The McIlvaine buffer solutions of pH4.0, pH4.6, pH5.0, pH5.6, pH5.8, pH6.0, pH6.6, pH7.0 are used in buffer solution (phosphate citrate buffer).After adding the 10mM flavones DMSO solution of 10 μ L in 88 μ L of buffer solution and mixing, 2 μ L are added 10mM anthocyanidin aqueous solution and mix.Reaction solution is added in the black wall cuvette (Shimadzu Seisakusho Ltd.) of ultramicron, is passed through UV2450 (Shimadzu Seisakusho Ltd.) measures the absorption spectra of 400-700nm.As a result, maximum absorption wavelength is attached in 577nm when being pH5.6 Closely, acromion becomes 590-596nm, and show and be found that the blue chrysanthemum of the absorption in the acromion region of 600-620nm Fresh flower valve is identical absorption spectra pattern, becomes a little partially purple blue.For pH5.8~6.0 when maximum absorption wave be grown to 593nm~596nm, color development are blue.For pH6.6~7.0 when maximum absorption wavelength further to long wavelength side displacement (597- 598nm), the absorbance of 400-500nm is got higher, and color development is turquoise~blue-green.It is acid with pH4.0 is become from pH5.6 Degree is got higher, short wavelength side displacement of the maximum absorption wavelength to 577nm~568nm, while absorbance significantly reduces.In addition, reaction It is reddish violet that the color of liquid changes from blue.It can be seen from the above result that being suitble to B cyclohexanol glycosidation delphinidin type anthocyanidin and flavone sugar The pH conditions of blue color development are 5.6~6.0 or so caused by the total color of glycosides, are measured in the petal squeezeding juice of blue chrysanthemum PH ranging from same degrees.

[embodiment 8：Cause from B cyclohexanol glycosidation delphinidin type anthocyanidin be total to color flavone glycoside structure it is different to color development The influence brought]

Using by Cross TLC methods explicitly in the petal of blue chrysanthemum by with 3 ', 5 '-glycosylation delphinidin types flowers Green element coexists and expresses the copigment of blue and with the flavone glycoside with Lt7MG and Tr7MG different structures, investigate them to B The influence that the color development of cyclohexanol glycosidation delphinidin type anthocyanidin is brought.The ternatin being dissolved into distilled water is used for anthocyanidin C5.For flavone glycoside, will only the 7- glucosides of the hydroxylated apiolin in 4 ' position of B rings with 7- rutinosides, 4 ' position of B rings by hydroxyl Change, 3 ' positions are by the acacetin 7- rutinosides of methoxylation, the 7- glucose of the hydroxylated cyanidenon in 3 ' position of B rings and 4 ' positions Glycosides, 7- (6 "-malonyl) glucoside (Lt7MG) and 3 ', 7- diglucosides, 3 ' position of B rings, 4 ' positions, 5 ' positions are hydroxylated Tr7MG is dissolved in DMSO to use.The 10mM flavones DMSO solution of 10 μ L is added in the 88 μ L of acetate buffer solution of pH5.6 and is mixed After conjunction, 2 μ L of addition 10mM anthocyanidin aqueous solution, by anthocyanidin and flavones with 1：5 amount is than mixing.Ultramicron is added in reaction solution Black wall cuvette (Shimadzu Seisakusho Ltd.) measures the absorption spectra of 400-700nm by UV2450 (Shimadzu Seisakusho Ltd.).As a result, only The a length of 559nm of maximum absorption wave when ternatin C5 is dissolved, and shows the whole for testing and being total to color with ternatin C5 The a length of 572-574nm of maximum absorption wave of flavone glycoside, the spectral pattern that acromion is 595-597nm, to express blue (figure 5).It can be seen from this result that in the difference to embellish a design caused by the hydroxyl and methoxyl group of 3 ' position of flavones B rings and 5 ' positions, 7 hydroxyls The difference of saccharide residue, the presence or absence of modification caused by the malonyl of 7 glucosyl groups can't be to blue table caused by total color Up to bringing big difference.It is therefore contemplated that 3 ', 5 '-glycosylation delphinidin type anthocyans whole in the petal of blue chrysanthemum Color is total to express blue with whole flavone glycosides.In addition, showing even if making the flavone glycoside different structure with chrysanthemum The other plant kind that is synthesized in spending of flavone glycoside in, by making 3 ', 5 '-glycosylation delphinidin type anthocyanidin accumulations also can table Up to blue.

[embodiment 9：The difference of anthocyanidin B rings to embellish a design is to the influence that color development is brought caused by total color]

Investigation Lt7MG expresses the structure feature of the anthocyanidin of blue by total color when coexisting.Fish pelargonium is used in anthocyanidin Pigment 3- (6 "-malonyl) glucoside (Pg3MG), Cy3MG, Dp3MG3 ' G, ternatin C5 (TC5).Such as the left institutes of Fig. 6 Show, in the hydroxylated Cy3MG of the Pg3MG, 3 ' positions that the 3 ' position of B rings of anthocyanidin and 5 ' positions are not modified, being unable to get color development is The collection of illustrative plates of blue.Obtained the maximum absorption is substantially the same in there is no the Dp3MG3 ' G of 5 ' the position glucosyl groups of the TC5 of expression blue, But the absorbance until acromion of 600nm to 620nm or so is low, shows on the contrary considerable near the 530nm of short wavelength side The collection of illustrative plates in acromion region is observed, to which the color development of blue will not be caused.Thus it shows as using flavone glycoside as copigment Anthocyanidin structure, the hydroxyl of 3 ' positions and 5 ' positions is by glycosylation particularly important.

[embodiment 10：The presence or absence of malonyl of 3 glucose of 3 ', 5 '-glycosylation delphinidin type anthocyanidin is to total color The influence that caused color development is brought]

When investigation is crosslinked with flavone glycoside, the malonyl of 3 glucose of 3 ', 5 '-glycosylation delphinidin type anthocyanidin has Whether nothing influences blue color development.Ternatin C5 (TC5) and preternatin C5 (pTC5) are used in anthocyanidin, auxiliary Lt7MG, Tr7MG are used in the flavone glycoside of pigment.Preternatin C5 are purified from the blue petal of chrysanthemum and are passed through LC- MS/MS is used after confirming structure.By anthocyanidin and flavones with 1：5 amount is than mixing.Add in the 88 μ L of acetate buffer solution of pH5.6 After adding 10 μ L of 10mM flavones DMSO solution to be mixed, 2 μ L of addition 10mM anthocyanidin aqueous solution are simultaneously mixed.Reaction solution is added to In the black wall cuvette (Shimadzu Seisakusho Ltd.) of ultramicron, the absorption spectra of 400-700nm is measured by UV2450 (Shimadzu Seisakusho Ltd.).Its As a result as shown in the right sides Fig. 6, even if when there is no malonyl on 3 glucose of anthocyanidin (Lt7MG+pTC5), with certain feelings Condition (Lt7MG+TC5) similarly shows the spectral pattern near maximum absorption wavelength 572nm, acromion 595nm, and color development is indigo plant Color.In addition, pH from 5.6 become 5.8 when, maximum absorption wavelength is displaced to 594nm from 572nm, to color development be blue.Another party It is faded in the mixed solution of pTC5 significantly fast, compared with the TC5 with malonyl in order to realize more stable blue in face Color development, it is believed that needs are modified by the malonyl in 3 glucose of anthocyanidin.

[embodiment 11：Hair caused by total color of 3 ', the 5 '-glycosylation delphinidin type anthocyanidin with C- glucosyl flavones Color]

The influence that bonding pattern sugared in the flavone glycoside of copigment brings blue expression is glycosylated into flavones using C- (Funakoshi) it is investigated.Ternatin C5 are used in 3 ', 5 '-glycosylation anthocyanidin, using passing through in copigment O- keys and sugar be bonded to flavones aglycone Lt7MG, by C- keys sugar be bonded to flavones aglycone reseda Plain 8-C- glucosides (orientin, Lt8CG), cyanidenon 6-C- glucosides (equal orientin, homo-orientin, Lt6CG), celery 5 kinds of dish element 8-C- glucosides (Vitexin, Ap8CG), apiolin 6-C- glucosides (isovitexin, Ap6CG).By cyanine Element is with flavones with 1：5 amount is than mixing.10 μ L of 10mM flavones DMSO solution are added in the 88 μ L of acetate buffer solution of pH5.6 and are mixed After conjunction, 2 μ L of addition 10mM anthocyanidin aqueous solution are simultaneously mixed.Reaction solution is added to the black wall cuvette (Shimadzu Seisakusho Ltd.) of ultramicron In, the absorption spectra of 400-700nm is measured by UV2450 (Shimadzu Seisakusho Ltd.).The results are shown in Figure 7 for it, is carried out in 8 sugar In the Lt8CG and Ap8CG of C- bondings, with Lt7OMG compared with absorption maximum and acromion to short wavelength side displacement number nm, from into For hyacinthine.On the other hand, 6 sugar carried out C- bonding Lt6CG and Ap6CG in, compared with Lt7OMG absorption maximum to Long wavelength side displacement 3nm or so, and then the absorbance of the acromion near 595nm also rises.Especially, in Ap6CG (different Vitex negundo var cannabifolias Element) in occur peak at 595nm, with Lt7OMG or others C- glycosyl flavones compared with expression stabilization and beautiful blue.It is tied Fruit makes the flavone glycoside for having carried out C bondings in 6 glucose coexist and shows for 3 ', 5 '-glycosylation delphinidin type anthocyanidin Blue expression it is effective.

[embodiment 12：Color development caused by the total color of 3 ', 5 '-glycosylation delphinidin type anthocyanidin and flavonol glycosides]

There to be a large amount of report flavonol glycosides accumulated simultaneously with anthocyanidin in petal same as flavone glycoside to be used as copigment The influence brought is expressed to blue caused by total color to investigate.Ternatin C5 are used in 3 ', 5 '-glycosylation anthocyanidin, Used as the Kaempferol 3- glucosides (Km3G) of flavonol glycosides, Quercetin 3- glucosides (Qu3G), Mongolian oak in copigment The Lt7MG of skin element 3- malonyls glucoside (Qu3MG) and Quercetin 3- rutinosides (rutin, Qu3RG) and flavones.It will spend Green element is with flavonols or flavones with 1：5 amount is than mixing.10mM flavonols DMSO is added in the 88 μ L of acetate buffer solution of pH5.6 Solution or 10 μ L of flavones DMSO solution and after mixing, 2 μ L of addition 10mM anthocyanidin aqueous solution are simultaneously mixed.Reaction solution is added to super In micro black wall cuvette (Shimadzu Seisakusho Ltd.), the absorption spectra of 400-700nm is measured by UV2450 (Shimadzu Seisakusho Ltd.).It is tied Fruit has the suction of acromion as shown in figure 8, showing a length of 572nm of maximum absorption wave in the Lt7MG of flavones near -595nm It receives spectrogram case and expresses blue.On the other hand, in Km3G, Qu3MG, Qu3RG of flavonols absorption maximum to 568-569nm's Short wavelength side displacement, acromion also near 592mm, become partially purple blue compared with Lt7MG.4 kind flavones of the Qu3G in investigation Become closest to using the Lt7MG of flavones as the absorption spectra of copigment, the position substantially phase of maximum absorption wavelength and acromion in alcohol Together.It is prompted by result above, is not only flavonoids, flavonols are by the way that with 3 ', 5 '-glycosylation delphinidin type anthocyanidin are total to color Can color development be blue, hyacinthine.

Claims

1. a kind of method, for the production method of the plant with blue series pattern, which is characterized in that make anthocyanidin B rings 3 ' and 5 ' positions are coexisted by glycosylation delphinidin type anthocyanidin and flavone glycoside or flavonol glycosides in the intracellular of plant.

2. according to the method described in claim 1, it is characterized in that, the flavone glycoside is selected from cyanidenon glucosides, pentahydroxy- Huang Ketoside, apiolin glucosides, acacetin glucosides and combination thereof.

3. according to the method described in claim 2, it is characterized in that, the cyanidenon glucosides is cyanidenon 7- malonyls Glucoside, cyanidenon 7- glucosides, cyanidenon 7,3 '-diglucoside, cyanidenon 8-C- glucosides, sweet-scented osmanthus Careless element 6-C- glucosides or their derivative.

4. according to the method described in claim 2, it is characterized in that, the quercetin glucosides is quercetin 7- malonyls Glucoside or derivatives thereof.

5. according to the method described in claim 2, it is characterized in that, the apiolin glucosides is apiolin 7- glucosides, celery Dish element 7- rutinosides, apiolin 8-C- glucosides, apiolin 6-C- glucosides or their derivative.

6. according to the method described in claim 2, it is characterized in that, the acacetin glucosides is acacetin 7- rutinosides Or derivatives thereof.

7. according to the method described in claim 1, it is characterized in that, the flavonol glycosides are selected from Kaempferol glucosides, Quercetin Glucosides and combination thereof.

8. the method according to the description of claim 7 is characterized in that the Kaempferol glucosides be Kaempferol 3- glucosides or its Derivative.

9. the method according to the description of claim 7 is characterized in that the quercetin glycoside is Quercetin 3- glucosides, Mongolian oak Skin element 3- (6 "-malonyl) glucoside, Quercetin 3- rutinosides or their derivative.

10. method according to claim 1 to 9, which is characterized in that 3 ' and 5 ' positions of the anthocyanidin B rings Delphinidin 3- (6 "-malonyl) glucoside -3 ' is selected from by glycosylation delphinidin type anthocyanidin, 5 '-diglucosides are Ternatin C5,3,3 ', 5 '-tri-glucose of delphinidin glycosides, that is, preternatin C5 and combination thereof.

11. according to method according to any one of claims 1 to 10, which is characterized in that 3 ' and 5 ' positions of the anthocyanidin B rings By glycosylation delphinidin type anthocyanidin and the flavone glycoside with 1：1~1：10 amount ratio coexists.

12. the method according to any one of claim 1~11, which is characterized in that the pH in the vacuole of the plant is 5.2~6.4.

13. the method according to any one of claim 1~12, which is characterized in that the plant is rose, lily, health It is fragrant, dahlia, iris or chrysanthemum.

14. a kind of plant or its from growing or he grows offspring, which is characterized in that be the plant with blue series pattern, in plant 3 ' and 5 ' positions of intracellular anthocyanidin B rings be total to flavone glycoside or flavonol glycosides by glycosylation delphinidin type anthocyanidin It deposits.

15. plant according to claim 14 or its from growing or he grows offspring, which is characterized in that the flavone glycoside choosing From cyanidenon glucosides, quercetin glucosides, apiolin glucosides, acacetin glucosides and combination thereof.

16. plant according to claim 15 or its from growing or he grows offspring, which is characterized in that the cyanidenon sugar Glycosides is cyanidenon 7- malonyls glucoside, cyanidenon 7- glucosides, cyanidenon 7,3 '-diglucoside, wood Rhinoceros grass element 8-C- glucosides, cyanidenon 6-C- glucosides or their derivative.

17. plant according to claim 15 or its from growing or he grows offspring, which is characterized in that the quercetin sugar Glycosides is quercetin 7- malonyl glucosides or derivatives thereof.

18. plant according to claim 15 or its from growing or he grows offspring, which is characterized in that the apiolin glucosides For apiolin 7- glucosides, apiolin 7- rutinosides, apiolin 8-C- glucosides, apiolin 6-C- glucosides or it Derivative.

19. plant according to claim 15 or its from growing or he grows offspring, which is characterized in that the acacetin sugar Glycosides is acacetin 7- rutinosides or derivatives thereof.

20. according to the method for claim 14, which is characterized in that the flavonol glycosides are selected from Kaempferol glucosides, quercitrin Plain glucosides and combination thereof.

21. according to the method for claim 20, which is characterized in that the Kaempferol glucosides be Kaempferol 3- glucosides or Its derivative.

22. according to the method for claim 20, which is characterized in that the quercetin glycoside be Quercetin 3- glucosides, Quercetin 3- (6 "-malonyl) glucoside, Quercetin 3- rutinosides or their derivative.

23. plant according to any one of claim 14~22 or its from growing or he grows offspring, which is characterized in that institute 3 ' and 5 ' positions for stating anthocyanidin B rings are selected from delphinidin 3- (6 "-malonyl) glucose by glycosylation delphinidin type anthocyanidin Glycosides -3 ', 5 '-diglucosides, that is, ternatin C5,3,3 ', 5 '-tri-glucose of delphinidin glycosides, that is, preternatinC5 and it Combination.

24. plant according to any one of claim 14~23 or its from growing or he grows offspring, which is characterized in that institute 3 ' and 5 ' positions of anthocyanidin B rings are stated by glycosylation delphinidin type anthocyanidin and the flavone glycoside or flavonol glycosides with 1： 1~1：10 amount ratio coexists.

25. plant according to any one of claim 14~24 or its from growing or he grows offspring, which is characterized in that institute It is 5.2~6.4 to state the pH in the vacuole of plant.

26. plant according to any one of claim 14~25 or its from growing or he grows offspring, which is characterized in that institute It is rose, lily, carnation, dahlia, iris or chrysanthemum to state plant.

27. a part for a kind of nutrition propagule, plant, tissue or cell, which is characterized in that from according to claim 14 Plant described in any one of~26 or its from growing or he grows offspring.

28. a kind of cut-flower or the processed goods made by the cut-flower, which is characterized in that from any one of claim 14~27 institute The plant stated or its from growing or he grows offspring.